Mixed xenogeneic chimerism is a promising approach to achieving tolerance across species barriers. This approach is being explored in nonhuman primates in Project 2. The studies in Project 3 will complement those in the nonhuman primate model by exploring the ability of porcine mixed xenogeneic chimerism to tolerize non-T cell components of the human immune system and analyzing the function of xenotolerant human T cells in detail. We have developed a humanized mouse model that allows generation of functional human T cells and B cells as well as human dendritic cells of several classes. These mice mount effective T cell responses and class-switched IgG responses following immunization and produce natural anti-pig xenoantibodies. We have also generated transgenic immunodeficient mice expressing porcine cytokines that promote porcine hematopoiesis from pig bone manow cells (BMCs). Using this model, we have demonstrated that the addition of porcine BMCs to the humanized mouse model results in central T cell tolerance induction specifically to the pig BMC donor, with preserved responsiveness to allogeneic and third party pig antigens. Our previous studies in the rat to mouse species combination have demonstrated that even low levels of mixed xenogeneic chimerism tolerize T cells centrally and tolerize natural killer (NK) cells and pre-existing natural antibody-producing B cells to the xenogeneic donor. In this proposal we will use this humanized mouse model to explore the ability of mixed porcine chimerism to tolerize human NK cells and B cells. We will also assess T cell function and the ability to clear opportunistic infections in pig-human mixed xenogeneic chimeras. We will: 1) Explore the ability of mixed xenogeneic chimerism to induce human NK cell tolerance in the pig-human species combination; 2) Explore the ability of mixed xenogeneic chimerism to induce human natural antibody-producing B cell tolerance in the pig-human species combination; and 3) Compare T cell immune responses in humanized mice with and without porcine mixed xenogeneic chimerism. These studies will assess T cell immune recognition in further detail and determine the ability to tolerize additional components of the human immune system that pose significant barriers to xenotransplantation. They have direct bearing on the mixed chimerism strategy being explored in the pig-->baboon model in Project 2 and will determine the potential utility of adding BMT to thymic transplantation in Project 1. The studies are also highly relevant to the non-Gal Nab studies in Projects 1 and 4.